Horseradish peroxidase hrp labeled goat anti rabbit secondary antibody

The second DRG and spinal dorsal horn sample was grounded on ice in an Radio Immunoprecipitation Assay (RIPA) lysis buffer. After centrifugation, the supernatant was collected to determine the protein concentration via Bicinchoninic Acid (BCA) methods. The sample was loaded for electrophoresis at constant voltages of 70 V and 90 V for stacking and resolving gels, respectively and then transferred to a Polyvinylidene Fluoride (PVDF) membrane at a constant current of 200 mA for 70 min. The membrane was blocked in 5% Bull Serum Albumin (BSA) at room temperature for 2 h. After the TBST wash (5 min, three times), the membrane was incubated in a bag with Tris Buffered saline Tween (TBST) -diluted antibody (1:100, Santa Cruz Biotechnology, USA) at 4°C overnight. After the second TBST wash (5 min, three times), the membrane was incubated with the Horseradish Peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (1:3000, Santa Cruz Biotechnology) at room temperature for 2 h. After the third TBST wash (10 min, three times), electrochemiluminescence (ECL) reagents were added to the membrane, and the DNR MicroChemi chemiluminescence gel imaging system was adopted for analysis. The relative gray value of the P2X₃ receptor band represented the relative expression level of the P2X₃ receptor.